O'Sullivan Dermot, Henry Michael, Joyce Helena, Walsh Naomi, Mc Auley Edel, Dowling Paul, Swan Niall, Moriarty Michael, Barnham Paul, Clynes Martin, Larkin Annemarie
National Institute for Cellular Biotechnology (NICB), Dublin City University, Glasnevin, Dublin 9, Ireland.
Tumour Biol. 2014 Jul;35(7):6983-97. doi: 10.1007/s13277-014-1857-5. Epub 2014 Apr 18.
Development of more effective therapeutic strategies for cancers of high unmet need requires the continued discovery of disease-specific protein targets for therapeutic antibody targeting. In order to identify novel proteins associated with cancer cell invasion/metastasis, we present here an alternative to antibody targeting of cell surface proteins with an established role in invasion; our functional antibody screening approach involves the isolation and selection of MAbs that are primarily screened for their ability to inhibit tumour invasion. A clonal population of the Mia PaCa-2, a pancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate MAbs with the objective of identifying membrane targets directly involved in cancer invasion. Selected MAb 7B7 can significantly reduce invasion in a dose-responsive manner in Mia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma cells. Using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis, the target antigen of anti-invasive antibody, 7B7, was determined to be the heterodimeric Ku antigen, Ku70/80, a core protein composed of the Ku70 and Ku80 subunits which is involved in non-homologous end-joining (NHEJ) DNA repair. RNA interference-mediated knockdown of Ku70 and Ku80 resulted in a marked decrease in the invasive capacity of Mia PaCa-2 clone 3 and DLKP-M cells, indicating that Ku70/Ku80 is functionally involved in pancreatic and lung cancer invasion. Immunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity in 37 PDAC tumours, indicating that this heterodimer is highly expressed in this aggressive cancer type. This study demonstrates that a functional MAb screening approach coupled with immunoprecipitation/proteomic analyses can be successfully applied to identify functional anti-invasive MAbs and potential novel targets for therapeutic antibody targeting.
开发针对需求未得到充分满足的癌症的更有效治疗策略，需要持续发现用于治疗性抗体靶向的疾病特异性蛋白质靶点。为了识别与癌细胞侵袭/转移相关的新蛋白质，我们在此提出一种替代方法，即针对在侵袭中起既定作用的细胞表面蛋白质进行抗体靶向；我们的功能性抗体筛选方法涉及分离和选择主要根据其抑制肿瘤侵袭能力进行筛选的单克隆抗体。使用具有高度侵袭性表型的胰腺导管腺癌（PDAC）细胞系Mia PaCa-2的克隆群体来产生单克隆抗体，目的是识别直接参与癌症侵袭的膜靶点。所选的单克隆抗体7B7可以以剂量反应方式显著降低Mia PaCa-2克隆3和DLKP-M肺鳞癌细胞的侵袭。通过免疫沉淀和液相色谱-串联质谱（LC-MS-MS）分析，确定抗侵袭抗体7B7的靶抗原为异二聚体Ku抗原Ku70/80，它是一种由Ku70和Ku80亚基组成的核心蛋白质，参与非同源末端连接（NHEJ）DNA修复。RNA干扰介导的Ku70和Ku80敲低导致Mia PaCa-2克隆3和DLKP-M细胞的侵袭能力显著下降，表明Ku70/Ku80在功能上参与胰腺癌和肺癌的侵袭。免疫组织化学分析显示37例PDAC肿瘤中有Ku70/Ku80免疫反应性，表明这种异二聚体在这种侵袭性癌症类型中高度表达。这项研究表明，功能性单克隆抗体筛选方法与免疫沉淀/蛋白质组学分析相结合，可以成功应用于识别功能性抗侵袭单克隆抗体和治疗性抗体靶向的潜在新靶点。
